Method for separation of monomeric polypeptides from aggregated polypeptides

• US 9,783,570 B2
• Filed: 06/28/2012
• Issued: 10/10/2017
• Est. Priority Date: 07/01/2011
• Status: Active Grant

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Baehner, Monika, Grossmann, Adelbert, Hepbildikler, Stefan
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US14/127,085
• Publication Number:
  US 20140142283A1
• Time in Patent Office: 1,930 Days
• CPC Class Codes: C07K 1/165 mixed-mode chromatography
• CPC Class Codes: C07K 1/18 Ion-exchange chromatography
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types
• CPC Class Codes: C07K 14/5443 IL-15
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

Herein is reported a method for obtaining a polypeptide in monomeric form comprising the steps of a) providing a solution comprising the polypeptide m monomeric form and m aggregated form, wherein the ratio of monomeric to aggregated form is 4:1 or less as determined by size exclusion chromatography, b) performing a mixed-mode chromatography in bind-and-elute mode, or a hydrophobic interaction chromatography in flow-through mode, or a size-exclusion chromatography, and c) performing a weak cation exchange chromatography in bind-and-elute mode or flow-through mode, and thereby obtaining the polypeptide m monomeric form.

Claims

1. A method for purifying a monomeric Fc-region containing polypeptide comprising the steps:

a) applying a solution comprising a Fc-region containing polypeptide in monomeric form and in aggregated form to a mixed-mode chromatography material, wherein the aggregated form is 20% or more as determined by size exclusion chromatography of the total Fc-region containing polypeptide in the solution, wherein said solution has a pH value of about 0.2 to about 2.5 pH units above the pl of the polypeptide, and recovering a solution comprising about 95% monomeric Fc-region containing polypeptide; and

b) applying the solution recovered in step a) to a weak cation exchange chromatography material, wherein said solution has pH value of about 0.2 to about 2.5 pH units above the pl of the polypeptide, and recovering the monomeric form of the Fc-region containing polypeptide.

2. The method according to claim 1, wherein the monomeric form of the Fc-region containing polypeptide is recovered from the flow-through of the weak cation exchange chromatography material of step b).

3. The method according to claim 1, wherein the solution applied to the mixed-mode chromatography material of step a) and/or the weak cation exchange chromatography material of step b) is buffered with a citrate buffer, acetate buffer, and/or MES buffer.